Daphnia Pulex Lineages in Response to Acute Copper Exposure Sneha Suresh1,2, Teresa J

Suresh et al. BMC Genomics (2020) 21:433 https://doi.org/10.1186/s12864-020-06831-4 RESEARCH ARTICLE Open Access Alternative splicing is highly variable among Daphnia pulex lineages in response to acute copper exposure Sneha Suresh1,2, Teresa J. Crease3, Melania E. Cristescu4 and Frédéric J. J. Chain1* Abstract Background: Despite being one of the primary mechanisms of gene expression regulation in eukaryotes, alternative splicing is often overlooked in ecotoxicogenomic studies. The process of alternative splicing facilitates the production of multiple mRNA isoforms from a single gene thereby greatly increasing the diversity of the transcriptome and proteome. This process can be important in enabling the organism to cope with stressful conditions. Accurate identification of splice sites using RNA sequencing requires alignment to independent exonic positions within the genome, presenting bioinformatic challenges, particularly when using short read data. Although technological advances allow for the detection of splicing patterns on a genome-wide scale, very little is known about the extent of intraspecies variation in splicing patterns, particularly in response to environmental stressors. In this study, we used RNA-sequencing to study the molecular responses to acute copper exposure in three lineages of Daphnia pulex by focusing on the contribution of alternative splicing in addition to gene expression responses. Results: By comparing the overall gene expression and splicing patterns among all 15 copper-exposed samples and 6 controls, we identified 588 differentially expressed (DE) genes and 16 differentially spliced (DS) genes. Most of the DS genes (13) were not found to be DE, suggesting unique transcriptional regulation in response to copper that went unnoticed with conventional DE analysis. To understand the influence of genetic background on gene expression and alternative splicing responses to Cu, each of the three lineages was analyzed separately. In contrast to the overall analysis, each lineage had a higher proportion of unique DS genes than DE genes suggesting that genetic background has a larger influence on DS than on DE. Gene Ontology analysis revealed that some pathways involved in stress response were jointly regulated by DS and DE genes while others were regulated by only transcription or only splicing. Conclusions: Our findings suggest an important role for alternative splicing in shaping transcriptome diversity in response to metal exposure in Daphnia, highlighting the importance of integrating splicing analyses with gene expression surveys to characterize molecular pathways in evolutionary and environmental studies. Keywords: Splicing, Copper, Metal pollution, Transcriptomics, Daphnia pulex, RNA-seq * Correspondence: [email protected] 1Department of Biological Sciences, University of Massachusetts Lowell, Lowell, MA 01854, USA Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Suresh et al. BMC Genomics (2020) 21:433 Page 2 of 14 Background transcript thereby regulating the amount of functional Anthropogenic activities such as mining and intensive transcript present in the cell [18]. agriculture have led to a substantial amount of heavy Identification of alternative splicing events from RNA- metal pollution in aquatic ecosystems [1]. Metal contam- seq data involves mapping reads to a reference genome ination poses a great threat to the overall health and sur- to identify splice junctions and counting the number of vival of aquatic organisms due to the long persistence reads aligning to particular exons and splice sites. Per- and bioaccumulation [2]. Exposure to environmental cent Spliced In (PSI) is a widely used metric for quanti- contaminants can induce genomic responses in organ- fying alternative splicing events and detecting differential isms affecting reproduction and survival [3]. In aquatic splicing between conditions. It represents the percentage invertebrates, exposure to metals such as copper has of transcripts including a particular exon or splice site been associated with increased production of reactive and is calculated from the read counts [19]. Unlike gene oxygen species, depletion of glutathione, inhibition of expression analysis where a read falling anywhere along oxidative phosphorylation and antioxidant systems, the gene will count towards expression, identification of DNA damage and inhibition of DNA repair mechanisms a splicing event requires the read to span the splice junc- [4]. Heavy metals further modulate the expression level tion and hence detection of differential splicing events of genes that are actively involved in protecting cells could potentially be biased towards more abundant tran- from metal-induced oxidative stress [2]. Recent genome- scripts [20]. Additionally, abundance of the final mRNA wide studies have provided important insights into the transcript is dependent on several factors such as cell molecular basis of transcription in response to metals, type, developmental stage, disease condition, the pres- but much less is known about the contribution of other ence of intronic and exonic enhancers and silencers, the mechanisms that regulate expression via RNA process- expression of various splicing factors, and can change in ing such as alternative splicing. response to external stimuli and cellular stress [21]. Alternative splicing is a regulatory process in eukary- Identification of intron retention events could be par- otes that generates multiple messenger RNAs (mRNAs) ticularly challenging as it can be difficult to distinguish from a single gene by selective removal or retention of true intron retention events from those arising from in- exons and introns from the pre-mRNA transcript [5]. It completely processed transcripts [22]. However, several is one of the primary mechanisms of gene expression computational tools have been developed for analysing regulation that contributes to transcriptional diversity alternative splicing events from RNA-seq data. While and activity in eukaryotes [6]. An extreme example is some tools like DEXSeq [23] rely on reads assigned to the Dscam gene that can generate 38,016 potential exons, others like JunctionSeq [24] and rMATS [25] use mRNA transcripts in Drosophila melanogaster and over reads aligned to both exons and splice junctions and can 13,000 potential mRNA isoforms in Daphnia via alterna- therefore identify differential splicing events even when tive splicing [7, 8]. Global analyses in eukaryotes have re- the exon expression level is consistent across different ported a large variation in the prevalence of alternative conditions [26]. splicing among taxa [9–11], from about 25% of genes in Several studies have reported that alternative splicing Caenorhabditis elegans [12] and 31% of genes in Dros- plays an important role in abiotic stress tolerance in ophila [13], to over 90% of genes in humans [14]. Gen- plants and mammals [27–30], but whether alternative omic architecture has been suggested to play a role in splicing is a common response to stress in aquatic inver- the diversity of observed frequencies of different types of tebrates is not well understood. Analyses of whole tran- alternative splicing [15]. The main types of alternative scriptomes of plants have shown that alternative splicing splicing events include exon skipping (ES), intron reten- regulates the expression level of genes involved in stress tion (IR), alternative 5′ splice site (A5SS), alternative 3′ response pathways and genes encoding the various com- splice site (A3SS) and mutually exclusive exons (MXE) ponents of a spliceosome, which is an RNA-protein [16]. Exon skipping is the most common type of alterna- complex that directs splicing of pre-mRNA transcripts tive splicing in animals, while intron retention events [31]. Recent studies on plants have also shown that abi- occur at high levels in plants, as well as most fungi and otic stresses such as exposure to high temperatures, high protists [11, 17]. Exon skipping, the presence of MXE, salinity or treatment with the plant hormone abscisic A3SS and A5SS within the exons lead to addition or re- acid alters the alternative splicing patterns of several moval of functional domains or changes in the amino or genes and promotes the use of non-canonical splice carboxy terminus of the protein product thereby affect- sites, thereby increasing the transcriptome diversity in ing its activity, localization, stability

Load more